PROJECT SUMMARY Innate immune-driven local inflammation response induced by ischemia-reperfusion injury (IRI) causes hepatic dysfunction and failure following liver transplantation. Mesenchymal stem cells (MSCs) has provided new insights into their potential clinical application, particularly for treating a variety of immune-mediated diseases. Previous studies have shown that crosstalk between MSCs and inflammatory cells is crucial in the mechanism of MSC-mediated immune modulation and tissue repair. Indeed, MSC-based therapy has been successfully applied in various human diseases. However, a number of clinical phase III trials of MSC immunotherapy failed to meet the primary endpoints because of the low efficacy of engrafted cells. Thus, increasing the immunosuppressive property of MSC by exploring novel immunoregulatory mechanisms emerges as one of the key challenges of MSC therapy. CD47 (integrin-associated protein, IAP) is a cell surface protein and expressed by virtually all cells in the body. CD47 expression is increased in hematopoietic stem cells (HSCs) after mobilization or induced inflammation. Ligation of CD47 can induce intracellular signaling resulting in cell differentiation and activation in response to stress. By binding to the cell surface glycoprotein, signal regulatory protein alpha (SIRP?), an inhibitory transmembrane receptor present on myeloid cells, CD47 can regulate cell function in the monocyte/macrophage lineage. Indeed, SIRP? is abundantly expressed in macrophages and has been implicated in regulating innate immunity during inflammatory response. SIRP? deficiency enhanced macrophage NF-?B activation and increased pro-inflammatory mediators, implying that SIRP? is an essential endogenous regulator of the innate immunity. A previous study has shown that Notch1 signaling regulates macrophage function and TLR4/NF-?B-driven inflammatory responses in IR-stressed mouse livers. It has also documented that Notch1 signaling alleviates the hepatocellular damage in orthotopic liver transplantation (OLT) in humans. Thus, CD47/SIRP? and Notch1 pathways are likely essential in the MSC-mediated regulation of innate immune responses in OLT. The overall goal of this proposal is to dissect the function of intracellular CD47/SIRP? signaling in MSC-mediated immune regulation and enhance MSC therapeutic efficacy by genetically modifying CD47/SIRP? in murine OLT. The hypothesis is that CD47/SIRP? signaling controls TLR4-driven inflammation in OLT recipients through activation of the Notch-Hes1 axis in MSC- mediated immune regulation. To test this hypothesis, the following specific aims are proposed: 1) Analyze the role of the CD47-SIRP? interaction in regulating TLR4-driven inflammatory response in MSC-mediated immune regulation in vitro; 2) Investigate the ability of immune regulation by CD47-modified MSC in vivo. These studies will increase understanding of CD47/SIRP?-mediated regulatory networks in MSC-mediated immunotherapy. Indeed, genetic modification of MSC to improve therapeutic efficacy may prove of paramount importance to effectively target IR stress in recipients of solid organ transplants.